Age-Related Macular Degeneration
Age-related macular degeneration (AMD) is a disease affecting the macular region of the eye, which is the area in the retina where the sharp vision is obtained. Macular degeneration is caused by the deterioration of the central portion of the retina, the inside back layer of the eye that records the images we see and sends them via the optic nerve from the eye to the brain. The retina's central portion, known as the macula, is responsible for focusing central vision in the eye, and it controls our ability to read, drive a car, recognize faces or colors, and see objects in fine detail.
AMD is the leading cause of irreversible blindness among the elderly in industrialized nations, and its prevalence increases in the population over the age of 60 (Klein et al., 1992; Mitchell et al., 1995). Numerous attempts have been made to understand the etiology of the disease, its pathophysiology and factors involved in the progression of the disease. A common early sign of AMD is the buildup of drusen, tiny yellow or white fat globules and extracellular material in the retina of the eye or on the optic nerve head. Drusen occurs as hard drusen (small, solid deposits that seem harmless) or larger deposits of soft drusen with indistinct borders. Soft drusen accumulating between the retinal pigment epithelium (RPE) and Bruch's membrane force these two structures apart.
Most people over 40 have a small amount of hard drusen, which can join to form soft drusen in AMD cases. However, not all soft drusen come from hard drusen.
There are two types of macular degeneration: the dry or atrophic type, and the wet or hemorrhagic type. The dry form of AMD, which constitutes 80% of all AMD patients, is characterized by the appearance of drusen. The presence of drusen is considered to be a pre-existing factor associated with the progression of the disease to either advanced dry AMD or wet AMD.
Alzheimer's Disease
Alzheimer's disease (AD) is an age-related progressive neurodegenerative disorder characterized by memory loss and severe cognitive decline (Hardy & Selkoe, 2002). The clinical features are manifested morphologically by excessive accumulation of extracellular aggregations of amyloid β-peptide (Aβ) in the form of amyloid plaques in the brain parenchyma, particularly in the hippocampus and cerebral cortex, leading to neuronal loss (Selkoe, 1991). In addition, in most mouse models of Alzheimer's disease the neurogenesis that normally occurs throughout life in the hippocampus of the adult brain is disrupted (Haughey et al., 2002). In Alzheimer patients, like in transgenic mice (PDGF-APPSw, Ind), some increase in neurogenesis takes place but is apparently not sufficient to overcome the disease (Jin et al., 2004a, b).
The Similarity Between AMD and Alzheimer's Disease
AMD and Alzheimer's disease are both chronic neurodegenerative disorders that affect a substantial proportion of elderly persons. Characteristic of these disorders is the irreversible loss of function, for which there is no cure. The degeneration occurring in AMD and Alzheimer's disease may, to some extent, have a common pathogenesis (Klayer et al., 1999). Although the etiology of both AMD and Alzheimer's disease is largely unknown, the pathogeneses of the two diseases show some striking similarities. In AMD, early histopathological manifestations are extracellular drusen deposits and basal laminar deposits (Hageman & Mullins, 1999). These lesions contain lipids, glycoproteins and glycosaminoglycans, which are presumably derived from a degenerating neuroretina (Kliffen et al., 1995). Accumulation of these deposits is associated with loss of photoreceptors and subsequent deterioration of macular function (Holz et al., 1994). As noted above, an early pathologic hallmark in Alzheimer's disease is the presence of extracellular senile plaques (Selkoe, 1991). These plaques are composed of many components, including small peptides generated by proteolytic cleavage of a family of transmembrane polypeptides known as amyloid precursor proteins. Two peptides that are widely regarded as major contributors to the pathology of Alzheimer's disease are known as amyloid-β (Aβ) peptides. Shared components of amyloid deposits and drusen include proteins such as vitronectin, amyloid P, apolipoprotein E, and even the Aβ peptides and amyloid oligomers that are associated with amyloid plaques in Alzheimer's disease (Luibl et al., 2006; Mullins et al., 2000; Yoshida et al., 2005).
The Aβ peptides present in Alzheimer's disease activate microglial cells to produce potentially neurotoxic substances such as reactive oxygen and nitrogen species, proinflammatory cytokines, complement proteins, and other inflammatory mediators that bring about neurodegenerative changes (Akiyama et al., 2000). The inflammatory response that has been associated with Alzheimer's disease often involves CD11b+ activated microglia, representing the innate arm of the immune system in the central nervous system (CNS) (Streit, 2004). CD11b+ microglia were reported to be associated with age-related normal human brain (Streit, 2004), and it is possible that such microglia are the ones that contribute both to age-related cognitive loss and to impaired neurogenesis (Monje et al., 2003). CD11b have also been found in patients with Alzheimer's disease (Akiyama & McGeer, 1990). Moreover, inflammatory mediators are present in amyloid deposits as well as in drusen, suggesting a possible common role for the inflammatory pathway in AMD and Alzheimer's disease (Hageman et al., 2001). A role for local inflammation in drusen biogenesis suggests that it is analogous to the process that occurs in Alzheimer's disease, where accumulation of extracellular plaques and deposits elicits a local chronic inflammatory response that exacerbates the effects of primary pathogenic stimuli (Akiyama et al., 2000).
Microglial Activation in Neurodegeneration
Microglia are bone marrow-derived glial cells, which are present within all layers of the adult human retina (Penfold et al., 1991). Several types are present which may be associated with neurons or with blood vessels, and some of these are antigen-presenting cells (APCs) (Penfold et al., 1991; Provis, 2001). The nature of microglial activation, either beneficial or harmful, in damaged neural tissue depends on how microglia interpret the threat (Butovsky et al., 2005). Although the presence of microglial cells in normal undamaged neural tissue has been debated for years, it is now an accepted fact (Nimmerjahn et al., 2005), including their presence in the eye. The role of microglia in inflammatory processes is controversial. On the one hand, participation of microglia in inflammatory process of the eye can stimulate mature retinal ganglion cells (RGCs) to regenerate their axons (Yin et al., 2003). On the other hand, the role of microglia in neurodegenerative processes may be detrimental to the neuronal tissue. Roque et al (1999) showed that microglial cells release soluble product(s) that induce degeneration of cultured photoreceptor cells. This controversy may be explained by the contradicting reports regarding the presence of antigen-presenting cells, which are crucial factors of an antigen-specific cell-mediated immune response. Immunological responses in neural retinal microglia are related to early pathogenic changes in retinal pigment epithelium pigmentation and drusen formation. Activated microglia may also be involved in rod cell death in AMD and late-onset retinal degeneration. A recent study has proposed that microglia, activated by primary rod cell death, migrate to the outer nuclear layer, remove rod cell debris and may kill adjacent cone photoreceptors (Gupta et al., 2003).
Like blood-derived macrophages, microglia exhibit scavenging of extracellular deposits, and phagocytosis of abnormal amyloid deposits in Alzheimer's disease. Such microglia, while efficiently acting as phagocytic cells, cause neuronal death by the secretion of mediators like tumor necrosis factor alpha (TNF-α) (Butovsky et al., 2005), and thus, while acting as phagocytic cells (Frenkel et al., 2005), they are apparently not efficient enough to fight off the Alzheimer's disease symptoms. In contrast to these resident microglia, microglia derived from the bone marrow of matched wild-type mice can effectively remove plaques (Simard et al., 2006). Moreover, an absence of normally functioning macrophages lead to the development of clinical AMD (Ambati et al., 2003). Thus, AMD, like Alzheimer's disease, illustrates a disease in which scavenging of abnormal deposits inevitably induces self-perpetuation of disease progression mediated by the phagocytic cell themselves (Gupta et al., 2003).
Protective Autoimmunity
Some years ago our group formulated the concept of ‘protective autoimmunity’ (Moalem et al., 1999). Both pro-inflammatory and anti-inflammatory cytokines were found to be critical components of a T cell-mediated beneficial autoimmune response, provided that the timing and the intensity of the T-cell activity was suitably controlled (Butovsky et al., 2005; Shaked et al., 2004), and depending on the nature of the disease (Schwartz et al., 2006). According to our concept, an uncontrolled autoimmunity leads to the commonly known condition of autoimmune diseases associated with overwhelmed activation of microglia (Butovsky et al., 2006a), as will be discussed below. The beneficial effect of the autoreactive T cells was found to be exerted via their ability to induce the CNS-resident microglia to adopt a phenotype capable of presenting antigens (Butovsky et al., 2001; Butovsky et al., 2005; Schwartz et al., 2006; Butovsky et al., 2006a; Shaked et al., 2004), expressing growth factors (Butovsky et al., 2005; Butovsky et al., 2006a;b), and buffering glutamate (Shaked et al., 2005).
In attempting to boost the efficacy of the protective autoreactive T cells, we tested many compounds in the search for a safe and suitable antigen for neuroprotection. We then suggested to use glatiramer acetate, also known as Copolymer 1 or Cop-1 (Kipnis et al., 2000; Avidan et al., 2004; Angelov et al., 2003), a synthetic 4-amino-acid copolymer known to be safe and currently used as a treatment for multiple sclerosis by a daily administration regimen (Copaxone®, Teva Pharmaceutical Industries Ltd., Israel). In our studies we have demonstrated its low-affinity cross-reaction with a wide range of CNS autoantigens. Because the affinity of cross-reaction is low, the Cop-1-activated T cells, after infiltrating the CNS, have the potential to become locally activated with little or no attendant risk of autoimmune disease (Kipnis et al., 2000).
A single injection of Cop-1 is protective in acute models of CNS insults (Kipnis et al., 2000; Avidan et al., 2004; Kipnis & Schwartz, 2002), while in chronic models occasional boosting is required for a long-lasting protective effect (Angelov et al., 2003). In the rat model of chronically high intraocular pressure, vaccination with Cop-1 significantly reduces RGC loss even if the pressure remains high. It should be noted that the vaccination does not prevent disease onset, but can slow down its progression by controlling the local extracellular environment of the nerve and retina, making it less hostile to neuronal survival and allowing the RGCs to be better able to withstand the stress (Schori et al., 2001; Benner et al., 2004; Kipnis & Schwartz, 2002; Kipnis et al., 2000).
For chronic conditions occasional boosting is needed. For example, in a model of chronically elevated intraocular pressure, weekly administration of adjuvant-free Cop-1 was found to result in neuroprotection (Bakalash et al., 2005). The neuroprotective effect of Cop-1 has been attributed in part to production of brain-derived neurotrophic factor (BDNF) (Kipnis et al., 2004b; Ziemssen et al., 2002).
Aggregated Aβ Induces Toxicity on Resident Microglia and Impairs Cell Renewal
Recent studies performed in our laboratory suggested that microglia exposed to aggregated Aβ, although effective in removing plaques, are toxic to neurons and impair neural cell renewal (Butovsky et al., 2006a); these effects are reminiscent of the response of microglia to invading microorganisms (as exemplified by their response to LPS) (Butovsky et al., 2005; Schwartz et al., 2006). Such activities are manifested by increased production of TNF-α, down-regulation of insulin-like growth factor (IGF-I), inhibition of the ability to express class II major histocompatibility complex (MHC-II) proteins and CD11c (a marker of dendritic cells) and thus to act as antigen-presenting cells (APCs), and failure to support neural tissue survival and renewal ((Butovsky et al., 2006a; Butovsky et al., 2005; Butovsky et al., 2006b). Further, we found that when microglia encounter aggregated β-amyloid, their ability to remove these aggregates without exerting toxic effects on neighboring neurons or impairing neurogenesis depends upon their undergoing a phenotype switch. A switch in microglial phenotype might take place via a local dialog between microglia and T-cells, which is mediated by T cell-derived cytokines such as interleukin (IL)-4. Addition of IL-4, a cytokine derived from T-helper (Th)-2 cells, to microglia activated by aggregated Aβ can reverse the down-regulation of IGF-I expression, the up-regulation of TNF-α expression, and the failure to act as APCs (Butovsky et al., 2005). The significance of microglia for in-vivo neural cell renewal was demonstrated by enhanced neurogenesis in the rat dentate gyrus after injection of IL-4-activated microglia intracerebroventricularly and by the presence of IGF-1-expressing microglia in the dentate gyms of rats kept in an enriched environment (Ziv et al., 2006). In rodents with acute or chronic EAE, injection of IL-4-activated microglia into the cerebrospinal fluid resulted in increased oligodendrogenesis in the spinal cord and improved clinical symptoms. The newly formed oligodendrocytes were spatially associated with microglia expressing MHC-II and IGF-I (Butovsky et al., 2006c).
In Both Alzheimer's Disease and AMD there are Systemic Components
Our first observation that systemic immune cells (in the form of T cells directed to certain self-antigens) can protect injured neurons from death came from studies in rodents showing that passive transfer of T cells specific to myelin basic protein reduces the loss of RGCs after a traumatic optic nerve injury (Moalem et al., 1999). We found that these T cells are also effective when directed to either cryptic or pathogenic epitopes of myelin basic protein, as well as to other myelin antigens or their epitopes (Mizrahi et al., 2002). These findings raised a number of critical questions. For example, are myelin antigens capable of protecting the nervous system from any type of acute or chronic insult? Is the observed neuroprotective activity of immune cells merely an anecdotal finding reflecting our experimental conditions, or does it point to the critical participation of the immune system in fighting off injurious conditions in the visual system and in the CNS in general? If the latter, does it mean that neurodegenerative diseases are systemic diseases? If so, can this finding be translated into a systemic therapy that would protect the brain, the eye, and the spinal cord?
In a series of experiments carried out over the last few years we have learned, firstly, that protective T cell response is a physiologically evoked response that might not be sufficient in severe insults or might not always be properly controlled. Moreover, we discovered that the specificity of such protective T cells depends on the site of the insult. Thus, for example, the protective effect of vaccination with myelin-associated antigens is restricted to injuries of the white matter, i.e., to myelinated axons (Mizrahi et al., 2002; Avidan et al., 2004; Schori et al., 2001). If the insult is to the retina, which contains no myelin, myelin antigens have no effect. Secondly, we observed that the injury-induced response of T cells reactive to specific self-antigens residing in the site of stress (eye or brain) is a spontaneous physiological response (Yoles et al., 2001). We then sought to identify the phenotype of the beneficial autoimmune T cells and to understand what determines the balance between a beneficial (neuroprotective) outcome of the T cell-mediated response to a CNS injury and a destructive effect causing autoimmune disease. We also examined ways of translating the beneficial response into a therapy for glaucoma. We found that in immune deficient animals the number of surviving RGCs following an insult in the eye, the spinal cord or the brain is significantly lower than in matched controls with an intact immune system, suggesting that the ability to withstand insult to the CNS depends on the integrity of the immune system and specifically on specific population within the immune system; those that recognize the site-specific self-antigens. Interestingly, the use of steroids caused significant loss of RGCs (Bakalash et al., 2003).
T Cells Specific to Antigens Residing in the Site of Damage Help Clean and Heal
In order to be protective, the anti-self T cells should home to the site of damage and be locally activated. This is why only those antigens that are being presented at the site of lesion can be used for the vaccination. Once activated, the T cells provide a source of cytokines and growth factors that shape the resident eye sentinels cells—the microglia, so as to make them active defensible cells that the eye can tolerate. Namely, such activated microglia can take up glutamate, remove debris and produce growth factors while refraining from production of agents that are part of their killing mechanism (e.g. TNF-α) to which the eye, like the brain, has a low tolerance (Butovsky et al., 2005; Butovsky et al., 2001; Barouch & Schwartz, 2002; Moalem et al., 2000; Shaked et al., 2005). Such T cells are constitutively controlled by physiologically existing regulatory T cells that are themselves amenable to control upon need (Kipnis et al., 2004a; Kipnis et al., 2002).
Reference is made to copending International Patent Application No. PCT/IL2007/000797 entitled “Activated myeloid cells for promoting tissue repair and detecting damaged tissue” filed by applicant at the Israel PCT Receiving Office (RO/IL) on the same date, the contents thereof being explicitly excluded from the scope of the present invention.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.